Factors influencing melanocyte pigment production and proliferation are complex and poorly understood, but some clues are suggested by changes that occur in both human melanocytes, and epidermal nevi (moles) during pregnancy. While it has been assumed that sex-steroid hormones influence these processes, the specific hormones, receptors, and downstream signaling pathways mediating any melanocyte effect are largely undefined. My preliminary results suggest that several pregnancy-associated sex steroid hormones significantly alter both melanocyte pigment production and cellular proliferation rate. My preliminary data indicates that estrogen and progesterone reciprocally regulate melanin production. Estrogen promotes melanin production, while progesterone decreases melanin production. The effects of these hormones are likely through nonclassical receptors rather than the classical steroid hormone receptors, which I have determined are not expressed in melanocytes. Additional preliminary findings suggest that the pregnancy associated progestin, allopregnanolone, increases proliferation and prevents the critical BRAF (V600E) growth-arrest, which is an essential step for the transition of benign nevus to melanoma. The experiments described in this proposal aim to 1: elucidate the signaling mechanisms through which estrogen and progesterone influence normal pigmentation, and 2: define the influence of allopregnanolone signaling on melanoma development, and identify the specific receptors and pathways through which allopregnanolone exerts its effect. In cultured melanocytes, depletion of GPER and PAQR7 using shRNA prevents the respective estrogen and progesterone effects. The goal of Aim IA is to determine the necessity and sufficiency of GPER and PAQR7 signaling to mediate the sex hormone effects in organotypic human tissue, and the goal of Aim IB is to determine the mechanisms through which GPER and PAQR7 influence melanin production. Allopregnanolone increases the proliferation rate and bypasses the BRAF (V600E) induced growth arrest in cultured melanocytes. Aim IIA is designed to determine mechanisms through which allopregnanolone promotes proliferation and growth-arrest bypass, and Aim IIB is designed to define the contribution of allopregnanolone signaling on melanoma development. My proposed work will determine the mechanisms through which pregnancy-associated sex steroid hormones influence melanocyte homeostasis, and may identify new therapeutic targets for pigmentation disorders and melanoma during pregnancy.